3D Structure and Function of CRAC Channels. The long-term objective of this proposal is to understand the mechanisms of ion permeation, ion selectivity, and gating in calcium release-activated calcium (CRAC) channels. CRAC channels play a central role in the immune response by generating the sustained influx of calcium (Ca2+) that is necessary for induction of T cell activation genes. CRAC channels, which are integral membrane proteins located in the plasma membrane, open in response to depletion of the Ca2+ stored within the endoplasmic reticulum (ER). Despite more than 20 years of research, the molecular components underlying this process of store-operated calcium entry (SOCE) were unknown. Recently, Orai protein was identified as the pore subunit and the STIM protein was determined to be the ER Ca2+ sensor. These long-awaited findings have accelerated research pertaining to the molecular mechanisms of CRAC channel function, revealing significant differences from other ion channels. Aside from being an integral membrane protein, Orai has no significant amino acid sequence homology to other known ions channels. The Orai channel is highly selective for Ca2+ ions, but this ion selectivity must be achieved by an architecture that is different from other Ca2+ channels. The channel also has an unprecedented mechanism of gating (the process that opens and closes the channel): the depletion of Ca2+ stored in the ER unites the Orai channel and its activator, STIM, despite their distinct membrane localizations. This study proposes to use X-ray crystallography to determine 3- dimensional structures of CRAC channels. Biochemical and biophysical techniques, including an assay to measure ion channel activity in vitro, will be used to correlate channel function with structural analysis. With these approaches we aim to: i) determine the 3-dimensional architecture of Orai, ii) investigate how Orai achieves high selectivity for Ca2+, and iii) study how the channel is gated by interactions with STIM. The proposed study will reveal basic principles of CRAC channel function, thereby making significant contributions to multiple fields of research including calcium signaling, ion channels, and membrane protein structural biology. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the architecture and molecular mechanisms of Orai, a calcium ion channel, at an atomic level of detail. Orai plays an important role in the immune system. A mutation of Orai is a cause of a lethal form of severe combined immunodeficiency in human patients. The proposed studies will provide a basis for the development of channel inhibitors that could be used to modulate immune responses. Recent data indicates that Orai channel function is also required for platelet activation and breast tumor cell migration, suggesting that channel inhibitors may have therapeutic applications for preventing arterial thrombosis, ischemic brain infarction, and breast cancer metastasis.